This study was done to compare meristem cultivation technique with shoot tip cultivation technique in order to obtain virus-free plants, as well as for comparison of micropropagation successes of two different nutrient mediums and for determining the effectiveness of real-time PCR for the detection of viruses. This experiment used two different types of garlic (Allium sativum & Allium tuncelianum), and two different media. Medium 2 proved to be more successful than Medium 1 in both A. tuncelianum (Kastamonu) and A. sativum (Kastamonu clone). Through real-time PCR analysis, in vitro plants were grown from shoot tip and meristem culture. We could not detect any virus in garlic plants grown via meristem cultivation. OYDV and LYSV viruses could be detected in shoot tip culture plants. OYDV virus was detected in 80% and 73%, respectively, of the tested plants for A. tuncelianum or A. sativum.
Garlic Virus-Free Production and Identification by Real-Time PCR
LYSV virus was detected in 67% of A. tuncelianum tested plants and 87% of A. sativum tested plants in this study. 18-20% of Turkey’s total garlic production comes from Taskopru in Kastamonu province. Kastamonu garlic (A. sativum clone) is suitable for winter consumption. It can be stored for a long period of time and can be processed due to its dry matter content . Tunceli garlic, also known as Allium tuncelianum, is found only in Tunceli province, Turkey. It is especially common around Munzur Mountains, Ovacik district. A. tuncelianum, unlike other garlics, has a single-cloved bulb and produces small, fertile seeds. Infected areas from pests and diseases transmitted via contaminated seedlings are the biggest problem in Turkey’s garlic production. Turkey does not separate garlic production areas from seedling production zones. Because of sexual sterility, contaminated garlic can carry diseases and pests.
This study had two objectives: (i) To compare meristem culture with shoot tip cultivation technique in order to obtain virus-free plants; (ii); to compare micropropagation success using two different nutrient media, and two different garlic species (A. tunceli anum & A. sativum); and (iii) To determine the effectiveness of real time PCR for detection of viruses. Materials and Methods
Between 2009 and 2010, this study was done at Adana Veterinary Control Institute (Turkey) and the Department of Horticulture University of Cukurova Turkey. Plant material was taken from Allium sativum (Kastamonu clone of garlic) and Allium tunelianum species of garlic that were found to have viruses. Kastamonu Taskopru and Tunceli Ovacik regions in Turkey provided samples of A. sativum, and A. tunceli anum garlic.
2.1. Meristem and Shoot Tip Meristem Culture Studies
- sativum, A. tuncelianum garlic varieties were propagated by meristem culture and shoot tip culture. For surface sterilisation, garlic cloves were placed in 25% sodium hypochlorite and left for 20 minutes. After that time, they were washed with sterile water 4-5 times. Sterile gloves were removed from their shoot tips using sterile forceps or scalpels. They were then placed in glass tubes containing MS nutrients medium . All cultures were placed in a growth chamber at 25°C under 3000 lux, 8 hours darkness and 16 hour light photoperiod conditions. For micropropagation, the shoot tips and germinated meristem were transferred to two different media: Medium 1: MS medium containing 0.25 mg L-1 2-IP and 0.2 mg NAA and 30g L-1 sucrose. Medium 2: MS medium containing 2 mg, 0.5, L-1 IBA and 30g L-1 sucrose. The number of shoots per plant was recorded after 6 weeks. After that, the shoots were subcultured. The experiment was carried out in an entirely random experimental design that included three replications and thirty plants per replication.
To evaluate the results, Variance analysis was performed. The Tukey test was used to control the significance of differences.
2.2. Real-Time PCR Studies
Sixty in vitro plant samples (15 from A. tunceli anum meristem and 15 from A. tunceli anum shoot tip Meristem Culture, A. tuncelianum) were used to conduct real-time PCR tests. For RNA extraction of garlic samples, High Pure Viral Nucleic Acid Kit was used. To a 1.5 mL microcentrifuge tube containing 100mg plant samples, 200 mL of working solution was added. 50 mL proteinaseK was also added. Pulverised. Mix the mixture and let it sit for 10 minutes at 72°C. Mixture was then completed by adding 100mL binding buffer. The mixture was then transferred to the upper reservoir of the combination of high pur filter tube and collection tube. It was spun in a centrifuge for 1 minute at 8,000g. The filter tube was then removed from the collection tube and placed in a new tube. Next, 500 mL of inhibitor removal buffer was added. Then the centrifuge was spun for 1 minute at 8,000 grams. The collection tube was replaced with a new one. Next, 500 mL of wash buffer was added. Then the centrifuge was spun for 1 minute at 10,000 g. The collecting tube was then changed and was washed again. The filter tube was then placed in a new 1.5 mL, nuclease-free microcentrifuge tub. 75 mL of elution buffer was added to the tube to remove viral nucleic acids. Finally, spin for 1 minute at 13,000g in a centrifuge. The filter tubes were then removed from the Eppendorf tubes and the purified viral nucleic acids extracted.
Pure viral nucleic acid was obtained and converted to cDNA using reverse transcription PCR (RT PCR). The samples were then incubated in a freezer at -15/-20degC. The Transcriptor High Fidelity cDNA synthesis Kit was used to convert RNA into cDNA. The process took two steps. To prepare the PCR mixture, 2 mL random primer, 1. mL of PCR-grade water and 8.4 mg of RNA per sample were used in the first step. The mixture was left to incubate at 65°C for 10 minutes. The second step contained 4 mL of transcriptor reverse transcription tampon, 0.25 mL protectorRNAse inhibitor and 2 mL deoxynucleotide mixture, 1 mL DTT and 1.1 mg transcriptor reverse transcriptase. Mix the two mixtures and let them incubate at 45-55degC (for 10-30 minutes) and 85degC (for 5 min). After cDNA synthesis was complete, it was stored at -20degC. Real-time real-time PCR was performed in a 20-mL volume that contained 1xSYBR green master mix (Roche applied sciences), 5 nM forward and reverse primers and PCR grade distilled water. The program consisted of: 10 minutes at 95degC to activate DNA polymerase; 45 cycles of 10 sec at 95degC; 30 sat 60degC; 1 sar 72degC; 30 sat 40degC; and 30 at 40degC. The thermocycler’s qualitative detection program was used to evaluate the mix. Mixture of 5 mL DNA extracted from samples, 2mL SYBERGreen master (it consisted of fast start Taq DNA Polymerase, reaction buffer and MgCl2, and SYBERGreen-Roche), 5mL forward primers and reverse primers, 2.5 mM Mg and PCR-grade distilled water. The mixture was used for real-time PCR. It required 600 cycles at 95degC to denature DNA. 45 cycles were performed at 10 s each at 60degC and 2 min at 72degC.